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Problem: In order to investigate the role of macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein -1 (MCP-1) in human ovulation, we studied the regulation of M-CSF and MCP-1 in cultured human granulosa cells., Method of Study: Immortalized granulosa cells (GC1a) were cultured in serum-free medium, and incubated with interleukin (IL)-1alpha, IL-1 receptor antagonist (ra) and tumor necrosis factor (TNF)-alpha. The supernatants were collected, and M-CSF and MCP-1 were measured by ELISA., Results: The levels of M-CSF and MCP-1 were increased after treatment with IL-1alpha (1 nm) and TNF-alpha (1 nm) in a time-dependent manner. The levels of M-CSF and MCP-1 were significantly increased after treatment with IL-1alpha and TNF-alpha in a dose-dependent manner. However, the levels of M-CSF and MCP-1 were significantly decreased by treatment with IL-1alpha (1 nm) and/or increasing concentrations of IL-1 ra., Conclusions: Our data indicated that M-CSF and MCP-1 were regulated by IL-1alpha and TNF-alpha. It was suggested that M-CSF and MCP-1 may play an important role in human preovulatory processes.

Purpose: HDAC inhibitors (HDACIs) have been shown to inhibit cancer cell proliferation, stimulate apoptosis, and induce cell cycle arrest. Our purpose was to investigate the antiproliferative effects of the HDACIs [suberoyl anilide bishydroxamine, valproic acid (VPA), trichostatin A, and sodium butyrate] against six endometrial cancer cell lines., Experimental Design: Endometrial cancer cells were treated with a variety of HDACIs, and the effect on cell growth, cell cycle, and apoptosis was measured. The ability of VPA to inhibit the growth of endometrial tumors growing in immunodeficient mice was also assessed., Results: Clonogenic assays showed that all cancer cell lines were sensitive to the growth inhibitory effect of HDACIs. Cell cycle analysis indicated that treatment with HDACIs decreased the proportion of cells in S phase and increased the proportion of cells in the G(0)-G(1) and/or G(2)-M phases of the cell cycle. Terminal deoxynucleotidyl transferase-mediated nick end labeling assays showed that HDACIs induced apoptosis. This was concomitant with altered expression of genes related to malignant phenotype, including an increase in p21(Waf1), p27(Kip7), and E-cadherin and a decrease in Bcl-2 and cyclin-D1 and -D2. Chromatin immunoprecipitation analysis revealed a remarkable increase in levels of acetylated histones associated with the p21 promoter after suberoyl anilide bishydroxamine treatment. In nude mice experiments, VPA inhibited significantly human uterine tumor growth without toxic side effects., Conclusions: These results suggest that HDACIs are effective in inhibiting growth of endometrial cancer cells in vitro and in nude mice, without toxic side effects. The findings raise the possibility that HDACIs may prove particularly effective in treatment of endometrial cancers.

Background: The aim of this study was to clarify changes in the expression of vascular endothelial growth factor (VEGF) during decidualization by endometrial stromal cells (ESCs) in vitro., Methods: ESCs were separated by enzymic digestion and filtration, and were cultured with RPMI 1640 in 10% fetal calf serum (FCS) and treated with medroxyprogesterone acetate (MPA) and dibutyryl-cyclic adenosine monophosphate (db-cAMP) to induce decidualization in vitro. The levels of prolactin (PRL) in the culture media were measured by enzyme immunoassay (EIA) and the levels of VEGF were measured by enzyme-linked immunosorbent assay (ELISA). The expression of VEGF mRNA by ESCs and decidualized cells was analyzed by Northern blot analysis., Results: In treated cells, PRL production was significantly increased due to treatment with both db-cAMP (0.5 mmol/L) and MPA (100 nmol/L). VEGF mRNA expression was detected without any stimulation by ESCs. VEGF production was also significantly greater in cells treated with db-cAMP (0.5 mmol/L) and MPA (1 nmol/L or 100 nmol/L) than in control cells. VEGF mRNA was also increased in association with decidualization in vitro., Conclusions: VEGF production increased in association with decidualization in this in vitro study. Decidualization of ESCs may play a role in the induction of growth in the human fetus and/or placentation. The mechanism involved may include influencing the production of angiogenic growth factors such as VEGF.

Poisonous snakebite is a rare complication of pregnancy. It has been suggested that snakebite poisoning during pregnancy may cause fetal loss. We report a case of intrauterine fetal death after a poisonous snakebite in the first trimester of pregnancy. A 29-year-old pregnant Japanese woman was admitted to hospital after being bitten by a pit viper on her right heel. The patient was at 10 weeks of gestation. In response to the bite, her right leg became extremely swollen, and paralysis of the right oculomotor nerve was observed. Intravenous administration of cepharanthine, ulinastatin, hydrocortisone sodium succinate, gabexate mesylate and antibiotics was started. Laboratory data suggested the presence of rhabdomyolysis. One week after admission, although she improved clinically and symptomatically, transvaginal ultrasonography revealed intrauterine fetal death. No vaginal bleeding was observed. A dilatation and curettage was performed. The patient made an uneventful recovery and was discharged from hospital 17 days after the snakebite. Consistent with other reports, in the first trimester intrauterine fetal death may especially occur when the mother has systemic symptoms, although its mechanism remains unclear., (Copyright 2004 S. Karger AG, Basel)

The characteristics and underlying mechanisms of new-onset ventricular arrhythmia during pregnancy have not been adequately investigated. We studied 11 pregnant women aged 35.4 +/- 3.1 years who experienced new-onset ventricular arrhythmias during pregnancy. The onset of their first episode of ventricular arrhythmia was distributed equally over the 3 trimesters. Ventricular premature contractions (VPCs) and ventricular tachycardia (VT) exhibited a monomorphic configuration in all patients; in 8 (73%) they originated from the right ventricular outflow tract. The frequency of VPCs decreased by more than 95% in 83% of the patients; couplets and VTs disappeared completely in all patients during the postpartum period. All parameters of heart rate variability were significantly lower during pregnancy than postpartum (P < .05). QT and QTc intervals were normal during pregnancy in all patients. The slope and the intersect of the QT/RR relationship obtained from Holter ECGs were not significantly different during pregnancy and in the postpartum period. New-onset ventricular arrhythmias in pregnancy manifest characteristics similar to those of idiopathic VT. Our findings suggest that various hemodynamic and neurohormonal changes associated with pregnancy play an important role in ventricular arrhythmogenesis.

To clarify the role of platelet-activating factor (PAF) in parturition, the effects of PAF on the secretion of PAF-acetylhydrolase (PAF-AH), a PAF-inactivating enzyme, by decidual macrophage populations were examined. The cells were isolated from human decidual tissue by enzymatic digestion, Ficoll-Paque centrifugation, or flow cytometric sorting. The nonhydrolyzable agonist of PAF, carbamyl-PAF (C-PAF), inhibited the secretion of PAF-AH by either decidual cells or flow cytometrically purified decidual macrophages. A specific PAF receptor antagonist, WEB 2086, blocked the C-PAF-induced inhibition. Lyso-PAF, a metabolite of PAF, had no effect on the enzyme secretion. An intracellular calcium channel blocker, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, partially blocked the inhibition by C-PAF, whereas extracellular calcium channel blockers, nifedipine and verapamil, were without effect. The inhibitory effect of C-PAF was also partially blocked by protein kinase C (PKC) inhibitors, sphingosine and H-7. A PKC activator, 12-O-tetradecanoylphobol 13-acetate, decreased the secretion of PAF-AH. The decrease was abolished by the addition of sphingosine and H-7. It is suggested that PAF inhibits the PAF-AH secretion by decidual macrophages and that the inhibitory action is mediated by a signal transduction mechanism involving intracellular calcium and PKC.

Background and Aims:  Platelet activating factor (PAF), a potent phospholipid mediator, has been implicated in a number of reproductive processes through ovulation to parturition. To clarify the regulatory mechanism of PAF metabolism in the decidua, we have investigated the effect of activation of protein kinase C (PKC) on the secretion of PAF-acetylhydrolase (PAF-AH), a PAF-inactivating enzyme, by human decidual macrophages. Methods:  Decidual macrophage populations were isolated from human decidua by using enzymic digestion, Ficoll-Paque centrifugation, or flow cytometric sorting. The cells were treated with a PKC activator (TPA), H-7, dibutyryl cyclic adenosine monophosphate (AMP), Bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra (acetoxymethyl)-ester (BAPTA/AM) and/or nifedipine. The activity of PAF-AH secreted in the culture medium was assayed. Results:  The PKC activator, TPA, inhibited the PAF-AH secretion by decidual cells in a dose-dependent manner. The TPA also decreased the enzyme secretion by flow cytometrically purified macrophages. The inhibitory effect of TPA was blocked by a PKC inhibitor, H-7. Protein kinase A (PKA) activation by dibutyryl cyclic AMP was without effect on the enzyme secretion. Calcium channel blockers, BAPTA/AM and nifedipine had no effect on the PAF-AH secretion. Conclusion:  It is suggested that the TPA-induced inhibition of PAF-AH secretion may be mediated, in part, by a PKC-dependent signal transduction, and that activation of PKC may result in the increase in the local concentration of PAF in the decidua because of its inhibitory effect on the PAF-AH secretion by decidual macrophages. (Reprod Med Biol 2003; 2 : 121-126).

Objective: To evaluate the effects of leptin on the production of interleukin (IL)-6 family cytokines and chemokines by human endometrial stromal cells (ESC) and epithelial cells., Design: The effects of leptin on the production of IL-6, IL-11, leukemia inhibitory factor (LIF), IL-8, growth-regulated oncogene (GRO)-alpha, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-3alpha by ESC and the endometrial epithelial cell line HHUA were investigated., Setting: Research laboratory at a medical university., Patient(s): Eight endometrial specimens in the late proliferative phase were used for the isolation of ESC., Intervention(s): ESC and HHUA were incubated for 24 hours with recombinant human leptin., Main Outcome Measure(s): The concentration of IL-6, IL-11, LIF, IL-8, GRO-alpha, MCP-1, and MIP-3alpha were measured using ELISAs., Result(s): Unstimulated ESC and HHUA constitutively secreted IL-6, IL-11, LIF, IL-8, GRO-alpha, MCP-1, and MIP-3alpha. The increase in levels of IL-6, IL-8, GRO-alpha, MCP-1, and MIP-3alpha in the culture media of ESC and HHUA paralleled the addition of increasing amounts of leptin. In contrast, the levels of IL-11 and LIF were not affected by leptin administration., Conclusion(s): The present findings suggest that leptin may be an additional modulator of IL-6 and chemokine expression in the endometrium. Leptin may contribute to the normal and pathological processes of human reproduction by the regulation of these cytokines in the local environment.

Elf-1 is a member of the Ets transcription factor family that regulates the genes involved in cellular growth and differentiation. Enhanced expression of Elf-1 has been reported in prostate cancer, breast cancer, and osteosarcoma. The aim of this study was to elucidate the involvement of Elf-1 in ovarian carcinogenesis. We analyzed serial frozen tissue sections from 26 patients with epithelial ovarian carcinoma and 10 patients with benign cystadenoma of ovary for Elf-1 protein expression, using fluorescent immunohistochemistry. We analyzed the relationship between the percentages of Elf-1-stained cells and patient characteristics, including histological classification, clinical stage, histological grade, clinical outcome, and survival rate. Elf-1 was weakly detected in some benign cystadenomas (0-5.5%). There was, however, abundant Elf-1 immunoreactivity in the nucleus of the ovarian carcinoma cells along with a little cytoplasmic staining. Scoring on the basis of the percentage of nuclear-positive cells indicated that nuclear Elf-1 expression was significantly associated with PCNA-labeling index (p=0.04), clinical stage (p<0.01), histological grade (p<0.01), and clinical outcome (p=0.02). However, there was no relationship between Elf-1 expression and histological classification. Survival data were available for all patients and demonstrated that Elf-1 expression was significantly associated with poor prognosis (p<0.01). Our results demonstrate that Elf-1 expression in ovarian carcinoma correlates with the malignant potential of this tumor.

Problem: Interleukin-8 (IL-8) and growth-regulated oncogene-alpha (GRO-alpha) have been proved to be important modulators of leukocyte chemotaxis in the mechanism of human ovulation. This study investigated the possible effects of IL-1alpha, tumor necrosis factor-alpha (TNF-alpha), protein kinase C (PKC) activators (TPA), and db-cyclic adenosine monophosphate (cAMP) on IL-8 and GRO-alpha production by immortalized GC1a and granulosa-lutein cells., Method of Study: Confluent granulosa-lutein cells were placed in serum-free medium before incubated for 8 hr with the above-mentioned test agents. Finally, we measured IL-8 and GRO-alpha levels in the culture media using an enzyme-linked immunosorbent assay (ELISA)., Results: Treatment of granulosa-lutein cells with of IL-1alpha (1 nM), TNF-alpha (1 nM), TPA (1 nM) and db-cAMP (100 microM) produced higher levels of IL-8 than untreated cells by 8 hr (2274.7 +/- 146.3, 1489.8 +/- 190.1, 1452.9 +/- 152.7, 1313.6 +/- 48.4 pg/mL, respectively; control = 457.7 +/- 38.2 pg/mL; P < 0.001). Treatment of granulosa-lutein cells with 1 nM of IL-1alpha, TNFalpha, TPA, and db-cAMP (100 microM) resulted in higher levels of GRO-alpha than untreated cells by 8 hr (993.7 +/- 9.5, 171.4 +/- 6.5, 147.5 +/- 6.7, 472.4 +/- 16.2 pg/mL respectively; control = 73.8 +/- 8.2 pg/mL; P < 0.001)., Conclusions: Our data strongly suggests roles for IL-1alpha, TNFalpha, and PKC activators in the inflammation-like mechanism of human ovulation. Furthermore, our study suggests a positive, but still debatable, role for cAMP in the same mechanism.

It has been demonstrated that human endometrial epithelial cells (EEC) and stromal cells (ESC) produce a variety of chemokines in vivo and in vitro. Growth-regulated oncogene (GRO)beta, which belongs to the CXC chemokine family, is a potent chemoattractant for neutrophils. To evaluate the regulation of GRO beta expression in the endometrium, the production of GRO beta by an EEC line, HHUA, and cultured ESC stimulated with various inflammatory mediators was examined by using an enzyme-linked immunosorbent assay. Unstimulated HHUA and ESC constitutively secreted GRO beta. Interleukin-1 beta, tumor necrosis factor-alpha and interferon-gamma significantly stimulated the expression of GRO beta by HHUA and ESC. Lipopolysaccharide also stimulated the expression of GRO beta by ESC, but not by HHUA. It is suggested that, in the human endometrium, the regulation of GRO beta expression is distinct from that of other CXC chemokines expressed in the endometrium, such as GRO alpha and interleukin-8. The modulation of the GRO beta concentration in the endometrium by inflammatory mediators may contribute to the normal and pathological processes of human reproduction by regulating the trafficking of neutrophils into the endometrium.

Objectives: Elf-1 is a member of the Ets transcription factor family that regulates the genes involved in cellular growth and differentiation. Enhanced expression of Elf-1 has been reported in prostate cancer, breast cancer, and osteosarcoma., Methods: To elucidate the involvement of Elf-1 in endometrial carcinogenesis, we analyzed serial frozen tissue sections from 31 patients with endometrial carcinoma and 20 patients with normal endometria for Elf-1 protein expression, using fluorescent immunohistochemistry. We analyzed the relationship between the percentages of Elf-1-stained cells and patient characteristics, including clinical stage, histological grade, presence of invasion to greater than one-half the myometrium, clinical outcome, and survival rate., Results: Elf-1 was weakly detected in some normal endometria in the proliferative phase (0-18.9%) and other normal endometria in the secretory phase (0-28.5%). There was, however, abundant Elf-1 immunoreactivity in the nucleus of the endometrial carcinoma cells along with a little cytoplasmic staining. Scoring on the basis of the percentage of nuclear-positive cells indicated that nuclear Elf-1 expression was significantly associated with PCNA-labeling index, clinical stage, histological grade, the presence of invasion to greater than one-half the myometrium, and clinical outcome (P < 0.01, respectively). Survival data were available for all patients and demonstrated that Elf-1 expression was significantly associated with poor prognosis (P < 0.01)., Conclusions: Our results demonstrate that Elf-1 expression in endometrial carcinoma correlates with the malignant potential of this tumor.

Objective: The aim is to clarify the role of platelet-activating factor and colony-stimulating factors in term and preterm parturition., Study Design: Decidual macrophage populations were obtained by an enzymic digestion, Ficoll-Paque centrifugation, or flow cytometric sorting. The effects of colony-stimulating factors on the platelet-activating factor acetylhydrolase secretion by these cells were examined., Results: Granulocyte-macrophage colony-stimulating factor inhibited the platelet-activating factor acetylhydrolase secretion by decidual macrophages. Granulocyte colony-stimulating factor also decreased the enzyme secretion but at higher concentrations than those required for granulocyte colony-stimulating factor. In contrast, macrophage colony-stimulating factor increased the enzyme secretion. These colony-stimulating factor-induced effects were specifically neutralized by the corresponding antibodies., Conclusion: Colony-stimulating factors may modulate the local concentration of platelet-activating factor in the decidua via their inhibitory or stimulatory effect on the secretion of platelet-activating factor acetylhydrolase, contributing to the regulation of term and preterm parturition at the maternal-fetal interface.

Survivin is a new member of the inhibitor of apoptosis family of anti-apoptotic proteins. It has been reported that survivin is expressed during fetal development and in cancer tissues. Because suppression of apoptosis is important for carcinogenesis and tumor growth, we investigated the expression of survivin in human endometrial carcinomas. We analyzed serial frozen sections for survivin protein expression in 26 patients with ovarian epithelial carcinoma and 10 patients with benign cystadenoma of the ovary by fluorescent immunohistochemistry. We analyzed the relationship between the percentages of survivin-stained cells and the characteristics of the patient including histological classification, clinical stage, histological grade, and clinical outcome. Survivin was weakly detected in some benign ovarian cystadenomas (0-12.1%). There was, however, abundant survivin immunoreactivity in the nucleus and/or cytoplasm of the epithelial ovarian carcinoma cells. Scoring on the basis of the percentage of positive cells indicated that survivin expression was significantly associated with PCNA-labeling index, clinical stage, histological grade, clinical outcome, and survival rate (p<0.01, respectively). We conclude that the survivin protein is a defining diagnostic marker for epithelial ovarian carcinomas that may also yield prognostic information.

Human endometrial stromal cells (ESC) can produce a variety of chemokines, especially after inflammatory stimulation. Interferon-gamma-inducible protein-10 (IP-10) is a potent chemoattractant for lymphocytes, and belongs to the family of non-ELR CXC chemokines. The expression of IP-10 in ESC after stimulation with interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), or lipopolysaccharide (LPS) was evaluated using an enzyme-linked immunosorbent assay and Northern blot analysis. A small amount of IP-10 protein was detected in the culture media of unstimulated ESC. The expression of IP-10 mRNA was detected in ESC. IFN-gamma, IL-1 beta, TNF-alpha and LPS significantly stimulated the expression of IP-10 mRNA and protein in ESC. These results suggest that the production of IP-10 by ESC is regulated by inflammatory mediators. The modulation of IP-10 concentrations in the local environment may contribute to the normal and pathological processes of human reproduction by regulating leukocyte trafficking in the endometrium.